Hisashi Nagase

The Relative Contribution of IL-4 Receptor Signaling and IL-10 to Susceptibility to Leishmania major

The roles of IL-10 and IL-4 receptor signaling were evaluated in a murine model of Leishmania major infection. In previous studies the L. major substrain LV39 caused progressive, nonhealing lesions in BALB/c mice deficient for IL-4R α-chain (IL-4Rα), while substrain IR173 was highly controlled. To explore whether IL-10 is responsible for inducing susceptibility to LV39, wild-type and IL-4Rα−/− mice were treated with anti-IL-10R mAb, and in a genetic approach, the IL-4Rα−/− mice were crossed with BALB/c IL-10−/− mice. In contrast to the lack of resistance conferred by IL-4Rα gene deletion, partial resistance to LV39 was conferred by IL-10 gene deletion or treatment of BALB/c mice with anti-IL-10R mAb. Lesion sizes and LV39 parasite numbers were further and dramatically reduced in both anti-IL-10R-treated IL-4Rα−/− mice and IL-4Rα × IL-10 double knockouts. Anti-IL-10R mAb treatment further suppressed parasite growth in IL-4Rα−/− mice infected with L. major IR173. Production of IFN-γ was only increased relative to wild-type or littermate controls in IL-4Rα−/− mice with complementary defects in IL-10. Comparisons of IFN-γ-treated infected macrophages in vitro indicated that LV39 required 25- to 500-fold greater concentrations of IFN-γ than IR173-infected macrophages to achieve a similar efficiency of parasite killing. These studies suggest that regardless of parasite substrain, IL-10 is as important as IL-4/IL-13 in promoting susceptibility to L. major and even more so for those substrains that are relatively resistant to IFN-γ mediated killing.

The intracellular domain of amyloid precursor protein induces neuron-specific apoptosis

Although amyloid precursor protein (APP) has central roles in Alzheimers disease, the physiological functions of this protein have yet to be fully elucidated. APP homologues show significant sequence conservation in the intracellular domain through evolution, which may reflect the functional importance of the intracellular domain of APP (AICD). To examine this possibility, we established embryonic carcinoma P19 cell lines overexpressing AICD. Although neurons could be differentiated from these cell lines with retinoic acid treatment, overexpression of AICD gave rise to neuron-specific cell death. Furthermore, DNA fragmentation was detected and TUNEL-positive cells were also Tuj1-positive neurons. Taken together, we concluded that AICD can induce neuron-specific apoptosis.

Despite Increased CD4+Foxp3+ Cells within the Infection Site, BALB/c IL-4 Receptor-Deficient Mice Reveal CD4+Foxp3-Negative T Cells as a Source of IL-10 in Leishmania major Susceptibility

BALB/c IL-4Rα−/− mice, despite the absence of IL-4/IL-13 signaling and potent Th2 responses, remain highly susceptible to Leishmania major substain LV39 due exclusively to residual levels of IL-10. To address the contribution of CD4+CD25+ T regulatory (Treg) cells to IL-10-mediated susceptibility, we depleted CD4+CD25+ cells in vivo and reconstituted IL-4Rα × RAG2 recipients with purified CD4+CD25− T cells. Although anti-CD25 mAb treatment significantly decreased parasite numbers in IL-4Rα−/− mice, treatment with anti-IL-10R mAb virtually eliminated L. major parasites in both footpad and dermal infection sites. In addition, IL-4Rα × RAG2 mice reconstituted with CD4+ cells depleted of CD25+ Treg cells remained highly susceptible to infection. Analysis of L. major-infected BALB/c and IL-4Rα−/− inflammatory sites revealed that the majority of IL-10 was secreted by the CD4+Foxp3− population, with a fraction of IL-10 coming from CD4+Foxp3+ Treg cells. All T cell IFN-γ production was also derived from the CD4+Foxp3− population. Nevertheless, the IL-4Rα−/−-infected ear dermis, but not draining lymph nodes, consistently displayed 1.5- to 2-fold greater percentages of CD4+CD25+ and CD4+Foxp3+ Treg cells compared with the BALB/c-infected dermis. Thus, CD4+Foxp3− T cells are a major source of IL-10 that disrupts IFN-γ activity in L. major-susceptible BALB/c mice. However, the increase in CD4+Foxp3+ T cells within the IL-4Rα−/− dermis implies a possible IL-10-independent role for Treg cells within the infection site, and may indicate a novel immune escape mechanism used by L. major parasites in the absence of IL-4/IL-13 signaling.

γ-Secretase-Regulated Signaling Pathways, such as Notch Signaling, Mediate the Differentiation of Hematopoietic Stem Cells, Development of the Immune System, and Peripheral Immune Responses

Notch signaling mediates the fates of numerous cells in both invertebrates and vertebrates. In the immune system, Notch signalling contributes to the generation of hematopoietic stem cells (HSCs), the promotion of HSC self-renewal, T lineage commitment, intrathymic T cell development, and peripheral lymphocyte differentiation/activation. The intracellular domain (ICD) of Notch is released from the cell membrane by γ-secretase and translocates to the nucleus to modulate gene expression. Hence, γ-secretase plays a central role in the regulation of Notch signaling. More than five dozen type 1 transmembrane proteins, including amyloid precursor protein, Notch, and Delta, are substrates for γ-secretase and their ICDs are released from the cell membrane. Therefore, it is highly possible that mechanisms similar to Notch signaling may widely contribute to γ-secretase-regulated signaling. Besides Notch, some transmembrane proteins such as CD44 and CSF-1R, which are important for immune responses, have been reported as substrates for γ-secretase. Since the ICDs of these proteins are also released by γ-secretase from the cell membrane and localize to the nucleus, it is thought that these ICDs modulate gene expression. Thus, γ-secretase-regulated signaling, including Notch signaling, may play a wide range of roles in the immune system.

The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis

Although amyloid precursor protein (APP) plays a central role in Alzheimers disease, the physiological functions of this protein have yet to be fully elucidated. As previously reported, we established an embryonic carcinoma P19 cell line expressing the intracellular domain of APP (AICD). While neurons were differentiated from these cell lines with retinoic acid treatment, expression of AICD induced neuron-specific apoptosis. As the first step to identify the genes involved in this process, we evaluated AICD-induced changes in gene expression through cell death. The levels of expression of 41,256 transcripts were monitored by DNA microarray analysis. The expression of 277 genes showed up-regulation by more than 10-fold in the presence of AICD. Conversely, the expression of 341 genes showed down-regulation to less than one-tenth of the original level. Reverse transcription-polymerase chain reaction of 17 selected genes showed excellent agreement with the microarray results. These results suggest that AICD induces dynamic changes in gene expression, which may be closely correlated with AICD-induced neuron-specific apoptosis.

Similar Mechanisms Regulated by γ-Secretase are Involved in Both Directions of the Bi-Directional Notch-Delta Signaling Pathway as well as Play a Potential Role in Signaling Events Involving Type 1 Transmembrane Proteins

In the canonical Notch signaling pathway, intramembrane cleavage by gamma-secretase serves to release an intracellular domain of Notch that has activity in the nucleus through binding to transcription factors. In addition, we showed that Notch also supplies signals to Delta, a major Notch ligand, to release the intracellular domain of Delta by gamma-secretase from the cell membrane, which then translocates to the nucleus, where it mediates the transcription of specific genes. Therefore, the Notch-Delta signaling pathway is bi-directional and similar mechanisms regulated by gamma-secretase are involved in both directions. Recently, it was demonstrated that many type 1 transmembrane proteins including Notch, Delta and amyloid precursor protein (APP) are substrates for gamma-secretase and release intracellular domains of these proteins from cell membranes. These observations that the common enzyme, gamma-secretase, modulates proteolysis and the turnover of possible signaling molecules have led to the attractive hypothesis that mechanisms similar to the Notch-Delta signaling pathway may widely contribute to gamma-secretase-regulated signaling pathways, including APP signaling which leads to Alzheimers disease. Here, we review the molecular mechanisms of the Notch-Delta signaling pathway in a bi-directional manner, and discuss the recent progress in understanding the biology of gamma-secretase-regulated signaling with respect to neurodegeneration.

γ-Secretase-Regulated Mechanisms Similar to Notch Signaling May Play a Role in Signaling Events, Including APP Signaling, Which Leads to Alzheimer’s Disease

Although γ-secretase was first identified as a protease that cleaves amyloid precursor protein (APP) within the transmembrane domain, thus producing Aβ peptides that are thought to be pathogenic in Alzheimer’s disease (AD), its physiological functions have not been fully elucidated. In the canonical Notch signaling pathway, intramembrane cleavage by γ-secretase serves to release an intracellular domain of Notch that shows activity in the nucleus through binding to transcription factors. Many type 1 transmembrane proteins, including Notch, Delta, and APP, have recently been shown to be substrates for γ-secretase, and their intracellular domains are released from the cell membrane following cleavage by γ-secretase. The common enzyme γ-secretase modulates proteolysis and the turnover of possible signaling molecules, which has led to the attractive hypothesis that mechanisms similar to Notch signaling contribute widely to proteolysis-regulated signaling pathways. APP is also likely to have a signaling mechanism, although the physiological functions of APP have not been elucidated. Indeed, we have shown that the intracellular domain of APP alters gene expression and induces neuron-specific apoptosis. These results suggest that APP signaling responds to the onset of AD. Here, we review the possibility of γ-secretase-regulated signaling, including APP signaling, which leads to AD.

γ-Secretase-Regulated Signaling Typified by Notch Signaling in the Immune System

Notch signaling mediates the fates of numerous cells not only in the nervous system but also in the immune system. Notch signaling contributes to the generation and maintenance of hematopoietic stem cells, lymphocyte development, and several immune responses. The molecular mechanism of Notch signaling is unique: ligands bind to the extracellular domain of Notch and trigger sequential proteolytic cleavages. Finally, γ-secretase releases the intracellular domain (ICD) of Notch (NICD) from the cell membrane, and NICD translocates to the nucleus. In the nucleus, NICD binds to transcription factors and modifies the expression of certain genes. Thus,γ-secretase controls Notch signaling. Recently, many type 1 transmembrane proteins have been reported to be substrates for γ-secretase, and their ICDs are released from the cell membrane to the cytoplasm. It has also been reported that ICDs of several of these substrates also translocate to the nucleus. These phenomena closely resemble that of Notch signaling. Therefore, the common enzyme -secretase controls the proteolysis and turnover of possible signaling molecules, which has led to the hypothesis that mechanisms similar to Notch signaling contribute widely to γ-secretase-regulated signaling pathways. Indeed, we have shown that the ICD of amyloid precursor protein (APP) alters gene expression and induces neuron-specific apoptosis. These observations suggest the existence of APP signaling that is controlled by γ-secretase. It is also likely that γ-secretase-regulated signaling pathways, besides Notch signaling, play an essential role in the immune system. In fact, CD44, which is involved in hematopoiesis and lymphocyte homing, seems to have a γ-secretase-regulated signaling mechanism. In this review, we focus not only on Notch signaling but also on other γ-secretase-regulated signaling pathways in the immune system.

Immunological characterization of C3H mice congenic for Faslprcg, C3H/HeJ-Faslprcg/Faslprcg

Faslpr (lpr) and Faslprcg (lprcg ) are allelic mutations of the Fas gene that is involved in apoptosis or programmed cell death. Lpr greatly reduces the expression of functional Fas and lprcg expresses the death domain-disabled, non-functional Fas on the cell surface. C3H/HeJ mice congenic for lprcg (C3H-lprcg ) were established and compared with C3H/HeJ-lpr/lpr (C3H-lpr) mice for their immunological and pathological features. Lymphadenopathy, splenomegaly, development of CD4-CD8-B220+ or double-negative (DN) T cells, renal pathology, and lymphoid cell infiltration in the lung and liver were not significantly different between C3H-lprcg and C3H-lpr mice. Noticeably, however, the production of serum immunoglobulin, autoantibodies against double-strand DNA and serum immune complexes were significantly lower in C3H-lprcg than in C3H-lpr mice. The results indicate that the death signal through the death domain of Fas is responsible for lymphoproliferation due to the accumulation of DN T cells and suggest that the region of Fas outside the death domain may be involved in autoantibody production. The newly-developed congenic C3H-lprcg mice will provide a powerful tool for research into the function of Fas apart from apoptosis.

HUMAN BRONCHIAL EPITHELIUM EXPRESSES INTERLEUKIN-9 RECEPTORS AND RELEASES NEUTROPHIL CHEMOTACTIC FACTOR

Growing evidence obtained from human genomic analysis and antigen-challenged transgenic mice suggests that interleukin-9 (IL-9) is a candidate factor in immunoglobulin E (IgE) production and thus is thought to be associated with bronchial inflammation and bronchial hyperresponsiveness (BHR). To evaluate the expression of the IL-9 receptor and its effect on the IL-9 human bronchial cell line BEAS-2B cells, reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemical investigation, and chemotaxis assay were performed. The components of the IL-9 receptor, consisting of IL-9 receptor α (CD129) and IL-2 receptor γ (CD132), were expressed on BEAS-2B cells as determined by RT-PCR and flow cytometry. BEAS-2B cells exposed to IL-9 released neutrophil chemotactic activity (NCA) in a time- and dose-dependent manner, and the presence of granulocyte colony-stimulating factor (G-CSF) was also detected. This factor is primarily involved in NCA for the measurement of cytokines and in the inhibition assay of neutrophil chemotaxis. These findings suggest that bronchial epithelial cells may express IL-9 receptors, and that IL-9 may induce airway inflammation through the release of G-CSF from bronchial epithelial cells.